Biomaterials (v.31, #14)

A series of biodegradable functional amino acid-based poly(ester amide)s (PEA-AG) were designed and synthesized by the solution co-polycondensation of amino acid (l-phenylalanine and dl-2-allylglycine) based monomers and dicarboxylic acid based monomers. Pendant carbon–carbon double bonds located in the dl-2-allylglycine were incorporated into these PEA-AGs, and the double bond contents could be adjusted by tuning the feed ratio of l-phenylalanine to dl-2-allylglycine monomers. Chemical structures of this new functional PEA-AG family were confirmed by FTIR and NMR spectra. The thermal properties of these polymers were investigated; increasing the methylene chain in both the amino acid and dicarboxlic acid segments resulted in a reduction in the polymer glass-transition temperature. The short-term in vitro biodegradation properties of PEA-AGs were investigated as a function of PEA-AG chemical structures and enzymes. Based on the weight loss data, PEA-AGs biodegraded much faster in an enzyme solution than in a PBS buffer solution. The utility of the pendant functional carbon–carbon double bonds in PEA-AG was demonstrated by synthesizing additional functional PEA derivatives. The incorporation of the functional pendant carbon–carbon double bonds along the PEA-AG chains could significantly expand the biomedical applications of these functional PEA-AGs via either their capability to conjugate bioactive agents or prepare additional useful functional derivatives.
Keywords: Poly(ester amide)s; Biodegradation; Double bond; Amino acid;

A platinum–chromium steel for cardiovascular stents by Barry J. O'Brien; Jon S. Stinson; Steve R. Larsen; Michael J. Eppihimer; William M. Carroll (3755-3761).
The desire to reduce the strut thickness of cardiovascular stents has driven the development of a new high strength radiopaque alloy, based on additions of platinum to a chromium-rich iron based matrix. This paper reports on initial development of the alloy and the rationale for selection of the composition. Data is presented for tensile and microstructural characterization, surface oxide analysis, corrosion resistance and endothelial cell response of the alloy. The results demonstrate the solid solution strengthening effect of the platinum, with an average yield strength of 480 MPa achieved. The material surface consists of primarily chromium oxide which contributes to the high corrosion resistance observed. The cell assay result suggests that surfaces of this Pt-enhanced alloy endothelialize in a manner comparable to stainless steel.
Keywords: Stent; Platinum; Chromium oxide; Endothelialization;

Neuronal adhesion and differentiation driven by nanoscale surface free-energy gradients by Guillaume Lamour; Ali Eftekhari-Bafrooei; Eric Borguet; Sylvie Souès; Ahmed Hamraoui (3762-3771).
Recent results indicate that, in addition to chemical, spatial and mechanical cues, substrate physical cues such as gradients in surface energy may also impact cell functions, such as neuronal differentiation of PC12 cells. However, it remains to be determined what surface effect is the most critical in triggering PC12 cell differentiation. Here we show that, beyond continuously probing the surface energy landscape of their environment, PC12 cells are highly sensitive to nanoscale chemical heterogeneities. Self-assembled monolayers of alkylsiloxanes on glass were used as a culture substrate. By changing the structure, ordering and chemical nature of the monolayer, the surface energy distribution is altered. While both well-ordered CH3 terminated substrates and bare glass (OH terminated) substrates did not favor PC12 cell adhesion, PC12 cells seeded on highly disordered CH3/OH substrates underwent enhanced adhesion and prompt neuritogenesis by 48 h of culture, without nerve growth factor treatment. These data illustrate that surface free-energy gradients, generated by nanoscale chemical heterogeneities, are critical to biological processes such as nerve regeneration on biomaterials.
Keywords: PC12 cells; Neuronal differentiation; Cell adhesion; Self-assembled monolayers (SAMs); Sum-frequency generation (SFG); Surface energy;

Thrombus formation is the major problem associated with biomaterials for blood contact medical devices. The immobilization of inhibitors to thrombin, a serine protease that plays a central role on the coagulation system, on the surface of biomaterials should be a good strategy to avoid blood clotting and increase their hemocompatibility. The aim of this work is the design of a nanostructured surface with capacity to adsorb and inactivate thrombin. The pentapeptide sequence d-Phenylalanine–Proline–Arginine–Proline–Glycine (fPRPG), that was described as a thrombin inhibitor, was immobilized onto tetra(ethylene glycol) terminated self-assembled monolayers (EG4-SAMs). Surface containing different amounts of fPRPG were prepared using different concentrations of N,N′-Carbonyldiimidazole (CDI) during immobilization. The efficiency of fPRPG immobilization was followed using ellipsometry, contact angle measurements, Infrared reflection absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy (XPS). Thrombin adsorption was quantified using radiolabelled thrombin and its activity in solution and after adsorption on the developed surfaces was assessed using a chromogenic assay. It was found that, although the immobilization of fPRPG on to EG4-SAMs does not increase its selectivity to thrombin, the activity of the adsorbed thrombin was inhibited in a peptide concentration dependent way. We concluded that SAMs with fPRPG immobilized in high amounts can be used as thrombin-inhibitor surfaces, which is a good step on the development of new surfaces for blood contact devices.
Keywords: Thrombin; Self-assembled monolayers; Surface functionalization; Peptide immobilization; Protein adsorption;

Surface structural conformations of fibrinogen polypeptides for improved biocompatibility by Mohammed Yaseen; Xiubo Zhao; Amy Freund; Alexander M. Seifalian; Jian R. Lu (3781-3792).
This work reports on how incorporation of silica nanocages into poly(urethane) copolymers (PU) affects conformational orientations of adsorbed fibrinogen and how different surfaces subsequently influenced HeLa cell attachment and proliferation. Incorporation of 2 wt% silica nanocages into poly(urethane) (PU4) substantially altered the surface topography of the films and some 50% of the surface was covered with the nanocages due to their preferential exposure. AFM studies revealed the deposition of a dense protein network on the soft polymeric domains of PU4 and much reduced fibrinogen adsorption on the hard nanocage domains. As on the bare SiO2 control surface, fibrinogen molecules adsorbed on top of the hard nanocages mainly took the dominant trinodular structures in monomeric and dimeric forms. In addition, net positively charged long α chains were prone to being hidden beneath the D domains whilst γ chains predominantly remained exposed. Dynamic interfacial adsorption as probed by spectroscopic ellipsometry revealed fast changes in interfacial conformation induced by electrostatic interactions between different segments of fibrinogen and the surface, consistent with the AFM imaging. On the PU surfaces without nanocage incorporation (PUA), however, adsorbed fibrinogen molecules formed beads-like chain networks, consistent with the structure featured on the soft PU4 domains, showing very different effects of surface chemical nature. Monoclonal antibodies specific to the α and γ chains showed reduced α but increased γ chain binding at the silicon oxide control and PU4 surfaces, whilst on the PUA, C18 and amine surfaces (organic surface controls) the opposite binding trend was detected with α chain binding dominant, showing different fibrinogen conformations. Cell attachment studies revealed differences in cell attachment and proliferation, consistent with the different polypeptide conformations on the two types of surfaces, showing a strong preference to the extent of exposure of γ chains.
Keywords: Biocompatibility; Protein adsorption; Fibrinogen; Cell adhesion; Polyurethane; Silica nanocage;

Human immune responses to porcine xenogeneic matrices and their extracellular matrix constituents in vitro by Alexandra Bayrak; Maria Tyralla; Juliane Ladhoff; Martina Schleicher; Ulrich A. Stock; Hans-Dieter Volk; Martina Seifert (3793-3803).
Several tissue engineering approaches for the treatment of cardiovascular diseases are based on a xenogeneic extracellular matrix. However, the application of engineered heart valves has failed in some patients, causing severe signs of inflammation by so far undetermined processes. Therefore we investigated the immune-mediated responses to porcine valve matrices (native, decellularized and glutaraldehyde-fixed) and to purified xenogeneic extracellular matrix proteins (ECMp). The induction of human immune responses in vitro was evaluated by analyzing the co-stimulatory effects of matrices and ECMp collagen and elastin on the proliferation of immune cell sub-populations via CFSE-based proliferation assays. The pattern of cytokine release was also determined. In porcine matrix punches we demonstrated strong immune responses with the native as well as the decellularized type, in contrast to attenuated effects with glutaraldehyde-fixed matrices. Furthermore, our results indicate that collagen type I (porcine and human) and human elastin were able to elicit proliferation in co-stimulation with anti-CD3 antibody, accompanied by a strong release of Th1 cytokines (IFN-γ, TNF-α). In contrast, porcine elastin did not elicit any response at all. This low immunogenic potential of porcine elastin suggests its suitability for the creation of new tissue engineering heart valve scaffolds in the future.
Keywords: Heart valve; Biocompatibility; Extracellular matrix; Cell proliferation; Inflammation;

Factors influencing osteoblast maturation on microgrooved titanium substrata by Myung-Hyun Lee; Namsik Oh; Suk-Won Lee; Richard Leesungbok; Sung-Eun Kim; Young-Pil Yun; Jong-Ho Kang (3804-3815).
In this study, we demonstrate surfaces with various dimensions of microgrooves fabricated by photolithography and subsequent acid etching that enhance various characteristics of titanium. Microgrooves with truncated V-shape in cross-section from 15 to 90 μm widths enabled us to report their exclusive effects on altering the surface chemistry and on enhancing the surface hydrophilicity, serum protein adsorption and osteoblast maturation on titanium substrata in a microgroove dimension-dependent manner. Further, acid etching and measurement direction separately affected the surface hydrophilicity results. By multiple correlation and regression analyses, surface chemistry, surface hydrophilicity and serum protein adsorption were determined to be the significant influential factors on osteoblast maturation. Within the limitations of this study, we conclude that combined submicron- and microtopography with relevant micro-dimension and structure enhance various characteristics of titanium, including surface hydrophilicity, which act as the essential factors influencing the osteoblast maturation on microgrooved titanium substrata.
Keywords: Surface topography; Surface analysis; Hydrophilicity; Protein adsorption; Osteoblast;

The biocompatibility of self-assembled brush polymers bearing glycine derivatives by Gahee Kim; Yecheol Rho; Samdae Park; Hyunchul Kim; Sejin Son; Heesoo Kim; Ik Jung Kim; Jung Ran Kim; Won Jong Kim; Moonhor Ree (3816-3826).
We have synthesized brush polymers with various glycine derivatives as the end groups of their long alkyl bristles. The polymers are thermally stable up to 170–210 °C and form good quality films through conventional spin- or dip-coating and subsequent drying. Interestingly, the thin films of these brush polymers exhibit different molecular multi-layer structures that arise through the efficient self-assembly of the bristles with glycine derivative end groups. These brush polymer films have hydrophilic surfaces and exhibit some water sorption. The extent of the water sorption by these films depends upon the nature of the glycine derivatives in the bristle end. These films not only repel fibrinogen molecules and platelets from their surfaces, but also have high resistance to bacterial adherence. Moreover, the films were found to provide conducive surface environments for the successful anchoring and growth of HEp-2 cells, and to exhibit excellent biocompatibility in mice. These brush polymers have potential uses in biomedical applications including medical devices, especially blood contacting devices such as catheters, stents, blood vessels, and biosensors, due to their enhanced biocompatibility and the reduced possibility of post-operative infection.
Keywords: Brush polymer; Glycine derivatives; Self-assembly; Bacterial adherence; Cell adhesion; Biocompatibility;

The enhanced characteristics of osteoblast adhesion to photofunctionalized nanoscale TiO2 layers on biomaterials surfaces by Tomohiko Miyauchi; Masahiro Yamada; Akiko Yamamoto; Fuminori Iwasa; Tetsuo Suzawa; Ryutaro Kamijo; Kazuyoshi Baba; Takahiro Ogawa (3827-3839).
Recently, UV photofunctionalization of titanium has been shown to be effective in enhancing osteogenic environment around this functional surface, in particular for the use of endosseous implants. However, the underlying mechanism remains unknown and its potential application to other tissue engineering materials has never been explored. We determined whether adhesion of a single osteoblast is enhanced on UV-treated nano-thin TiO2 layer with virtually no surface roughness or topographical features. Rat bone marrow-derived osteoblasts were cultured on UV-treated or untreated 200-nm thick TiO2 sputter-coated glass plates. After an incubation of 3 h, the mean critical shear force required to initiate detachment of a single osteoblast was determined to be 1280 ± 430 nN on UV-treated TiO2 surfaces, which was 2.5-fold greater than the force required on untreated TiO2 surfaces. The total energy required to complete the detachment was 37.0 ± 23.2 pJ on UV-treated surfaces, 3.5-fold greater than that required on untreated surfaces. Such substantial increases in single cell adhesion were also observed for osteoblasts cultured for 24 h. Osteoblasts on UV-treated TiO2 surfaces were larger and characterized with increased levels of vinculin expression and focal contact formation. However, the density of vinculin or focal contact was not influenced by UV treatment. In contrast, both total expression and density of actin fibers increased on UV-treated surfaces. Thin layer TiO2 coating and UV treatment of Co–Cr alloy and PTFE membrane synergistically resulted in a significant increase in the ability of cell attachment and osteoblastic production of alkaline phosphatase. These results indicated that the adhesive nature of a single osteoblast is substantially enhanced on UV-treated TiO2 surfaces, providing the first evidence showing that each individual cell attached to these surfaces is substantially more resistant to exogenous load potentially from blood and fluid flow and mechanical force in the initial stage of in vivo biological environment. This enhanced osteoblast adhesion was supported synergistically but disproportionately by enhancement in focal adhesion and cytoskeletal developments. Also, this study demonstrated that UV treatment is effective on nano-thin TiO2 depositioned onto non-Ti materials to enhance their bioactivity, providing a basis for TiO2-mediated photofunctionalization of biomaterials, a new method of developing functional biomaterials.
Keywords: Bone–titanium integration; Vinculin; Osseointegration; Actin; Titanium; UV;

Biomimetic hydrogels with pro-angiogenic properties by James J. Moon; Jennifer E. Saik; Ross A. Poché; Julia E. Leslie-Barbick; Soo-Hong Lee; April A. Smith; Mary E. Dickinson; Jennifer L. West (3840-3847).
To achieve the task of fabricating functional tissues, scaffold materials that can be sufficiently vascularized to mimic functionality and complexity of native tissues are yet to be developed. Here, we report development of synthetic, biomimetic hydrogels that allow the rapid formation of a stable and mature vascular network both in vitro and in vivo. Hydrogels were fabricated with integrin binding sites and protease-sensitive substrates to mimic the natural provisional extracellular matrices, and endothelial cells cultured in these hydrogels organized into stable, intricate networks of capillary-like structures. The resulting structures were further stabilized by recruitment of mesenchymal progenitor cells that differentiated into a smooth muscle cell lineage and deposited collagen IV and laminin in vitro. In addition, hydrogels transplanted into mouse corneas were infiltrated with host vasculature, resulting in extensive vascularization with functional blood vessels. These results indicate that these hydrogels may be useful for applications in basic biological research, tissue engineering, and regenerative medicine.
Keywords: Hydrogel; Endothelial cell; Smooth muscle cell; Mesenchcymal stem cell;

Human bone marrow mesenchymal stem cells (hBMSCs) require an invasive procedure to harvest, and have lower self-renewal potential with aging. Umbilical cord mesenchymal stem cells (hUCMSCs) are a relatively new stem cell source; this study reveals a self-setting and load-bearing calcium phosphate construct that encapsulates these stem cells. The flexural strength (mean ± sd; n  = 5) of the hUCMSC-encapsulating calcium phosphate cement (CPC) increased from (3.5 ± 1.1) MPa without polyglactin fibers, to (11.7 ± 2.1) MPa with 20% of polyglactin fibers (p  < 0.05). hUCMSCs attached to the bone mineral-mimicking scaffold in the osteogenic media and differentiated down the osteogenic lineage, yielding elevated alkaline phosphatase (ALP) and osteocalcin (OC) gene expressions. ALP and OC on the CPC-fiber scaffold was 2-fold those on CPC control without fibers. hUCMSCs encapsulated inside the scaffolds retained excellent viability and cell density. The encapsulated hUCMSCs inside four different constructs successfully differentiated down the osteogenic lineage and synthesized bone minerals, as confirmed by mineral staining, SEM, and XRD. The percentage of mineral area synthesized by the encapsulated hUCMSCs increased from about 3% at day-7, to 12% at day-21 (p  < 0.05). In conclusion, this study demonstrated that hUCMSCs encapsulated in the bioengineered scaffolds osteo-differentiated and synthesized bone minerals. The self-setting CPC–chitosan–fiber scaffold supported the viability and osteogenic differentiation of the encapsulated hUCMSCs, and had mechanical strength matching that of cancellous bone.
Keywords: Umbilical cord stem cells; Calcium phosphate cement scaffolds; Cell encapsulation; Osteogenic differentiation; Load-bearing; Bone tissue engineering;

Chondrogenic differentiation of human adult adipose-derived stem cells was studied in vitro for the development of engineered cartilage tissue. Cells cultured under dynamic conditions in polyglycolic acid (PGA) scaffolds produced substantially higher glycosaminoglycan (GAG) and total collagen levels than cells in pellet cultures. This result reflects the importance of cell attachment and cell–scaffold interactions in stem cell differentiation and chondrogenesis. Although gene expression levels for both aggrecan and collagen type II were up-regulated significantly in PGA cultures treated with transforming growth factor β1 (TGF-β1), synthesis of GAG but not collagen type II was enhanced in tissue constructs when TGF-β1 was added to the medium. Bone morphogenetic protein-6 (BMP-6) in the presence of TGF-β1 was effective in improving GAG and total collagen production when the cells were pre-treated with fibroblast growth factor-2 (FGF-2) prior to scaffold seeding. Extending the culture duration from 2 to 5 weeks did not improve cartilage development in PGA scaffolds; loss of cells from the constructs suggested that the rate of scaffold degradation exceeded the rate of replacement by ECM during the 5-week period. Stem cells in PGA scaffolds were cultured in perfusion-type recirculation bioreactors operated with periodic medium flow reversal. The highest levels of GAG and collagen type II accumulation were achieved in the bioreactor cultures after the seeding cell density was increased from 2 × 107 to 4 × 107 cells per scaffold.
Keywords: Bioreactor; Cartilage tissue engineering; ECM (extracellular matrix); Growth factors; Mesenchymal stem cell; Polyglycolic acid;

One of the major obstacles in drug discovery is the lack of in vitro three-dimensional (3D) models that can capture more complex features of a disease.Here we established a in vitro physiological model of the metabolic syndrome (MS) using cell-assembly technique (CAT), which can assemble cells into designated places to form complex 3D structures. Adipose-derived stromal (ADS) cells were assembled with gelatin/alginate/fibrinogen. Fibrin was employed as an effective material to regulate ADS cell differentiation and self-organization along with other methods. ADS cells differentiated into adipocytes and endothelial cells, meanwhile, the cells were induced to self-organize into an analogous tissue structure. Pancreatic islets were then deposited at designated locations and constituted the adipoinsular axis with adipocytes. Analysis of the factors involved in energy metabolism showed that this system could capture more pathological features of MS. Drugs known to have effects on MS showed accordant effects in this system, indicating that the model has potential in MS drug discovery. Overall, this study demonstrated that cell differentiation and self-organization can be regulated by techniques combined with CAT. The model presented could result in a better understanding of the pathogenesis of MS and the development of new technologies for drug discovery.
Keywords: Cell-assembly technique; Stem cell; Biomimetic material; Self-organize; Metabolic syndrome; Drug discovery;

The effect of two point mutations in GDF-5 on ectopic bone formation in a β-tricalciumphosphate scaffold by Philip Kasten; Ingo Beyen; Dirk Bormann; Reto Luginbühl; Frank Plöger; Wiltrud Richter (3878-3884).
The osteoinductivity of human growth-and-differentiation factor-5 (GDF-5) is well established, but a reduced amount of ectopic bone is formed compared to other members of the bone morphogenetic protein (BMP) family like BMP-2. We hypothesized that swap of two BMP-receptor-interacting residues of GDF-5 to amino acids present in BMP-2 (methionine to valine at the sites 453 and 456) may improve the bone formation capacity of the mutant GDF-5. Heterotopic bone formation of a mutant GDF-5 coated β-TCP carrier was compared to carriers coated with similar amounts (10 μg) of GDF-5 and BMP-2 in SCID mice. Four week explants revealed 6-fold higher ALP activity in the mutant GDF-5 versus the wild type GDF-5 group (p < 0.0001) and 1.4-fold higher levels compared to BMP-2 (p < 0.006). Bone area in histology was significantly higher in mutant GDF-5 versus all other groups at 4 weeks; however, at 8 weeks BMP-2 reached a similar neo-bone formation like mutant GDF-5. Micro-CT evaluation confirmed higher values in the mutant GDF-5 and BMP-2 groups compared to wild type GDF-5. In conclusion, the mutant GDF-5 showed superior bone formation capacity than GDF-5, and a faster induction at similar final outcome as BMP-2. Mutant GDF-5 thus represents a promising new GDF-5 variant for bone regeneration possibly acting via an increased binding affinity to the BMP-type I receptor.
Keywords: Growth factor; Mutation; GDF-5; Heterotopic bone formation; Osteogenic differentiation; Beta tricalcium phosphate ceramic;

An elastomeric patch derived from poly(glycerol sebacate) for delivery of embryonic stem cells to the heart by Qi-Zhi Chen; Hikaru Ishii; George A. Thouas; Alexander R. Lyon; Jamie S. Wright; Jonny J. Blaker; Wojciech Chrzanowski; Aldo R. Boccaccini; Nadire N. Ali; Jonathan C. Knowles; Siân E. Harding (3885-3893).
We hypothesize that a combinatorial approach of ventricle constraint and stem cell therapy would offer a greater benefit for the treatment of heart failure than either strategy alone. A heart patch would serve two therapeutic purposes: biomechanical support and cell delivery. In this study, we describe a hybrid heart patch engineered from a synthetic elastomer, poly(glycerol sebacate) (PGS), supplemented with cardiomyocytes differentiated from human embryonic stem cells (hESCs). In line with two therapeutically relevant considerations, i.e. biocompatibility and cell delivery efficiency, the PGS was (a) pre-conditioned in culture medium for 6 days, and (b) prepared without gelatin coatings to facilitate detachment and delivery of cardiomyocytes following patch implantation. Following pre-conditioning under physiological conditions, the PGS patch material without gelatin coating was found to satisfactorily support cardiomyocyte viability and attachment, with active cell beating for periods of longer than 3 months until interrupted. Dynamic culture studies revealed that cells detached more efficiently from the uncoated surface of PGS than from gelatin-coated PGS. No significant differences were detected between the beating rates of human embryonic stem cell-derived cardiomyocytes on tissue culture plate and the pre-conditioned and gelatin-uncoated PGS. PGS patches sutured over the left ventricle of rats in vivo remained intact over a 2 week period without any deleterious effects on ventricular function. We conclude that PGS is a suitable biomaterial for stem cell-based regeneration strategies to restore cardiomyocyte function, and the hybrid heart patch engineered under optimal conditions would be a promising support device for the cardiac repair.
Keywords: Poly(glycerol sebacate); Embryonic stem cell; In vitro; In vivo; Heart;

In vivo engineering of a functional tendon sheath in a hen model by Liang Xu; Dejun Cao; Wei Liu; Guangdong Zhou; Wen Jie Zhang; Yilin Cao (3894-3902).
Repair of injured tendon sheath remains a major challenge and this study explored the possibility of in vivo reconstruction of a tendon sheath with tendon sheath derived cells and polyglycolic acid (PGA) fibers in a Leghorn hen model. Total 55 Leghorn hens with a 1 cm tendon sheath defect created in the left middle toe of each animal were randomly assigned into: (1) experimental group (n  = 19) that received a cell-PGA construct; (2) scaffold control group (n  = 18) that received a cell-free PGA scaffold; (3) blank control group (n  = 18) with the defect untreated. Tendon sheath cells were isolated, in vitro expanded, and seeded onto PGA scaffolds. After in vitro culture for 7 days, the constructs were in vivo implanted to repair the sheath defects. Alcian blue staining confirmed the ability of cultured cells to produce specific matrices containing acidic carboxyl mucopolysaccharide (mainly hyaluronic acid). In addition, the engineered sheath formed a relatively mature structure at 12 weeks post-surgery, which was similar to that of native counterpart, including a smooth inner surface, a well-developed sheath histological structure with a clear space between the tendon and the engineered sheath. More importantly, Work of Flexion assay revealed that the tendons needed less power consumption to glide inside the engineered sheath when compared to the tendons which were surrounded by scar-repaired tissues, indicating that the engineered sheaths had gained the function to a certain extent of preventing tendon adhesion. Taken together, these results suggest that tendon sheaths that are functionally and structurally similar to native sheaths are possible to be engineered in vivo using tendon sheath cells and PGA scaffolds.
Keywords: Engineered tendon sheath; Tendon adhesion; Tendon sheath derived cells; Acidic carboxyl mucopolysaccharide; Work of flexion assay;

Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering by Nahoko Asakawa; Tatsuya Shimizu; Yukiko Tsuda; Sachiko Sekiya; Tadashi Sasagawa; Masayuki Yamato; Fumio Fukai; Teruo Okano (3903-3909).
Reconstructing a vascular network is a common task for three-dimensional (3-D) tissue engineering. Three-dimensional stratified tissues were created by stacking cell sheets, and the co-culture with endothelial cells (ECs) in the tissues was found to lead to in vitro pre-vascular network formation and promoted in vivo neovascularization after their transplantation. In this study, to clarify the effect of tissue fabrication process on a pre-vascular network formation, human origin ECs were introduced into the stratified tissue in several different ways, and the behavior of ECs in various positions of the 3-D tissue were compared each other. Human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), and their mixture were harvested as an intact cell sheet from temperature-responsive culture dish at low-temperature (<20 °C). Single mono-culture EC sheet was stacked with two NHDF-sheets in different orders, and 3 co-cultured cell sheets were layered by a cell sheet collecting device. Morphological analyses revealed that pre-vascular networks composing of HUVECs were formed in all the triple layer constructs. Confocal microscope observation showed that the pre-vascular networks formed tube structures like a native microvasculature. These data indicate that the primary EC positioning in 3-D tissues may be critical for vascular formation.
Keywords: Vascular; Temperature-responsive culture dish; Endothelial cell; Tissue engineering; Cell sheet; Tubular formation;

Intra-uterine tissue engineering of full-thickness skin defects in a fetal sheep model by Nynke A. Hosper; Alex J. Eggink; Luc A.J. Roelofs; Rene M.H. Wijnen; Marja J.A. van Luyn; Ruud A. Bank; Martin C. Harmsen; Paul J. Geutjes; Willeke F. Daamen; Toin H. van Kuppevelt; Dorien M. Tiemessen; Egbert Oosterwijk; Jane J. Crevels; Willeke A.M. Blokx; Fred K. Lotgering; Paul P. van den Berg; Wout F.J. Feitz (3910-3919).
In spina bifida the neural tube fails to close during the embryonic period and it is thought that prolonged exposure of the unprotected spinal cord to the amniotic fluid during pregnancy causes additional neural damage. Intra-uterine repair might protect the neural tissue from exposure to amniotic fluid and might reduce additional neural damage. Biodegradable collagen scaffolds may be useful in case of fetal therapy for spina bifida, but biochemical properties need to be studied. The aim of this study was to investigate whether biodegradable collagen scaffolds can be used to treat full-thickness fetal skin defects. We hypothesized that the pro-angiogenic growth factors VEGF and FGF2 would enhance vascularization, epidermialization and lead to improved wound healing. To investigate the effect of these two growth factors, a fetal sheep model for skin defects was used. Compared to wounds treated with bare collagen scaffolds, wounds treated with growth factor-loaded scaffolds showed excessive formation of capillaries and less myofibroblasts were present in these wounds, leading to less contraction. This study has demonstrated that collagen scaffolds can be used to treat fetal skin defects and that the combination of collagen scaffolds with VEGF and FGF2 had a beneficial effect on wound healing.
Keywords: Spina bifida; Fetal wound healing; Intra-uterine repair; Collagen scaffold; Vascular endothelial growth factor; Basic fibroblast growth factor;

A complex 3D human tissue culture system based on mammary stromal cells and silk scaffolds for modeling breast morphogenesis and function by Xiuli Wang; Lin Sun; Maricel V. Maffini; Ana Soto; Carlos Sonnenschein; David L. Kaplan (3920-3929).
Epithelial-stromal interactions play a crucial role in normal embryonic development and carcinogenesis of the human breast while the underlying mechanisms of these events remain poorly understood. To address this issue, we constructed a physiologically relevant, three-dimensional (3D) culture surrogate of complex human breast tissue that included a tri-culture system made up of human mammary epithelial cells (MCF10A), human fibroblasts and adipocytes, i.e., the two dominant breast stromal cell types, in a Matrigel™/collagen mixture on porous silk protein scaffolds. The presence of stromal cells inhibited MCF10A cell proliferation and induced both alveolar and ductal morphogenesis and enhanced casein expression. In contrast to the immature polarity exhibited by co-cultures with either fibroblasts or adipocytes, the alveolar structures formed by the tri-cultures exhibited proper polarity similar to that observed in breast tissue in vivo. Only alveolar structures with reverted polarity were observed in MCF10A monocultures. Consistent with their phenotypic appearance, more functional differentiation of epithelial cells was also observed in the tri-cultures, where casein α- and -β mRNA expression was significantly increased. This in vitro tri-culture breast tissue system sustained on silk scaffold effectively represents a more physiologically relevant 3D microenvironment for mammary epithelial cells and stromal cells than either co-cultures or monocultures. This experimental model provides an important first step for bioengineering an informative human breast tissue system, with which to study normal breast morphogenesis and neoplastic transformation.
Keywords: Silk; Epithelial cells; Fibroblasts; Adipocyte; Co-culture; Tri-culture;

The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation by Stephanie K. Seidlits; Zin Z. Khaing; Rebecca R. Petersen; Jonathan D. Nickels; Jennifer E. Vanscoy; Jason B. Shear; Christine E. Schmidt (3930-3940).
We report the ability to direct the differentiation pathway of neural progenitor cells (NPCs) within hydrogels having tunable mechanical properties. By modifying the polymeric sugar hyaluronic acid (HA), a major extracellular matrix component in the fetal mammalian brain, with varying numbers of photocrosslinkable methacrylate groups, hydrogels could be prepared with bulk compressive moduli spanning the threefold range measured for neonatal brain and adult spinal cord. Ventral midbrain-derived NPCs were photoencapsulated into HA hydrogels and remained viable after encapsulation. After three weeks, the majority of NPCs cultured in hydrogels with mechanical properties comparable to those of neonatal brain had differentiated into neurons (ß-III tubulin-positive), many of which had extended long, branched processes, indicative of a relatively mature phenotype. In contrast, NPCs within stiffer hydrogels, with mechanical properties comparable to those of adult brain, had differentiated into mostly astrocytes (glial fibrillary acidic protein (GFAP)-positive). Primary spinal astrocytes cultured in the hydrogel variants for two weeks acquired a spread and elongated morphology only in the stiffest hydrogels evaluated, with mechanical properties similar to adult tissue. Results demonstrate that the mechanical properties of these scaffolds can assert a defining influence on the differentiation of ventral midbrain-derived NPCs, which have strong clinical relevance because of their ability to mature into dopaminergic neurons of the substantia nigra, cells that idiopathically degenerate in individuals suffering from Parkinson's disease.
Keywords: Hydrogel; Hyaluronic acid/hyaluronan; Progenitor cell; Neural cell; Astrocyte;

Preparation and characterization of decellularized cornea using high-hydrostatic pressurization for corneal tissue engineering by Yoshihide Hashimoto; Seiichi Funamoto; Shuji Sasaki; Takako Honda; Shinya Hattori; Kwangwoo Nam; Tsuyoshi Kimura; Manabu Mochizuki; Toshiya Fujisato; Hisatoshi Kobayashi; Akio Kishida (3941-3948).
To prepare acellular corneal scaffold, we used high-hydrostatic pressurization (HHP) to decellularize porcine cornea. The HHP method disrupts cells by hydrostatic pressurization, and then the disrupted cells' components are removed by washing with a cell culture medium. Porcine corneas were hydrostatically pressed at 980 MPa at 10 or 30 °C for 10 min to make them opaque. There was no change in the thickness of the corneas immediately after the pressurization, but they swelled during the washing process. The cornea swelling caused by HHP was suppressed when medium containing 3.5% w/v dextran was used. For H-E staining of the cornea decellularized with the HHP method, the complete removal of corneal cells was confirmed. Furthermore, when the corneas were immersed in glycerol for 1 hour, their optical properties were restored to those of native corneas. In an animal study, when acellular porcine corneas were implanted into rabbit cornea, no immune reaction occurred and the turbid corneas became clear. The decellularized corneas obtained through HHP could be useful as a corneal scaffold for tissue regeneration.
Keywords: Cornea; ECM; Scaffold; Xenotransplantation;

The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro by Eileen Gentleman; Yann C. Fredholm; Gavin Jell; Nasrin Lotfibakhshaiesh; Matthew D. O'Donnell; Robert G. Hill; Molly M. Stevens (3949-3956).
Bioactive glasses (BG) which contain strontium have the potential to combine the known bone regenerative properties of BG with the anabolic and anti-catabolic effects of strontium cations. Here we created a BG series (SiO2–P2O5–Na2O–CaO) in which 0–100% of the calcium was substituted by strontium and tested their effects on osteoblasts and osteoclasts in vitro. We show that ions released from strontium-substituted BG enhance metabolic activity in osteoblasts. They also inhibit osteoclast activity by both reducing tartrate resistant acid phosphatase activity and inhibiting resorption of calcium phosphate films in a dose-dependent manner. Additionally, osteoblasts cultured in contact with BG show increased proliferation and alkaline phosphatase activity with increasing strontium substitution, while osteoclasts adopt typical resorption morphologies. These results suggest that similarly to the osteoporosis drug strontium ranelate, strontium-substituted BG may promote an anabolic effect on osteoblasts and an anti-catabolic effect on osteoclasts. These effects, when combined with the advantages of BG such as controlled ion release and delivery versatility, may make strontium-substituted BG an effective biomaterial choice for a range of bone regeneration therapies.
Keywords: Bioactive glass; Osteoblast; Osteoclast; Bone regeneration; Osteoporosis;

Enzymatically degradable poly(ethylene glycol) based hydrogels for adipose tissue engineering by Ferdinand P. Brandl; Anna K. Seitz; Jörg K.V. Teßmar; Torsten Blunk; Achim M. Göpferich (3957-3966).
Adipose tissue engineering requires biomaterials that promote the differentiation of seeded adipocytes. Here, we report on the development and characterization of in situ forming, poly(ethylene glycol) (PEG) based hydrogels for soft tissue augmentation. Branched PEG-amines were modified with collagenase-sensitive peptides and cross-linked with branched PEG-succinimidyl propionates without the use of free-radical initiators (enzymatically degradable hydrogels). Alanine-modified PEG-amines were used for the preparation of non-degradable gels. Depending on the used polymer concentration, the strength of degradable gels after swelling ranged from 1708 to 7412 Pa; the strength of non-degradable hydrogels varied between 1496 and 7686 Pa. Enzyme mediated gel degradation occurred within 10, 16, and 19 days (5%, 10%, and 15% initial polymer content). To evaluate their suitability as scaffold materials for adipose tissue engineering, the hydrogels were functionalized with the laminin-derived adhesion peptide YIGSR, and seeded with 3T3-L1 preadipocytes. Compared to a standard two-dimensional cell culture model, the developed hydrogels significantly enhanced the intracellular triglyceride accumulation of encapsulated adipocytes. Functionalization with YIGSR further enhanced lipid synthesis within differentiating adipocytes. Long-term studies suggested that enzymatically degradable hydrogels furthermore promote the formation of coherent adipose tissue-like structures featuring many mature unilocular fat cells.
Keywords: Adipose tissue engineering; Biomimetic material; Cell adhesion; Degradation; Hydrogel; Mechanical properties;

The behaviour of neural stem cells on polyhydroxyalkanoate nanofiber scaffolds by Xian-Yi Xu; Xiao-Tao Li; Si-Wu Peng; Jian-Feng Xiao; Chao Liu; Guo Fang; Kevin C. Chen; Guo-Qiang Chen (3967-3975).
Polyhydroxyalkanoates (PHA) have demonstrated their potentials as medical implant biomaterials. Neural stem cells (NSCs) grown on/in PHA scaffolds may be useful for repairing central nervous system (CNS) injury. To investigate this possibility, nanofiber matrices (scaffolds) prepared from several PHA via a novel phase separation process were studied to mimic natural extracellular matrix (ECM), and rat-derived NSCs grown in the PHA matrices were characterized regarding their in vitro differentiation behaviors. All three PHA materials including poly(3-hydroxybutyrate) (PHB), copolymer of 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB), and copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) supported NSC growth and differentiation both on their 2D films and 3D matrices. Among three PHA nanofiber matrices, PHBHHx one showed the strongest potentials to promote NSC differentiation into neurons which is beneficial for CNS repair. Compared to the 2D films, 3D nanofiber matrices appeared to be more suitable for NSC attachment, synaptic outgrowth and synaptogenesis. It was suggested that PHBHHx nanofiber scaffolds (matrices) that promote NSC growth and differentiation, can be developed for treating central nervous system injury.
Keywords: PHB; Polyhydroxyalkanoates; Nanofiber; Matrices; Scaffold; Neural stem cells;

Chitosan and collagen type I are naturally derived materials used as cell carriers because of their ability to mimic the extracellular environment and direct cell function. In this study beta-glycerophosphate (β-GP), an osteogenic medium supplement and a weak base, was used to simultaneously initiate gelation of pure chitosan, pure collagen, and chitosan-collagen composite materials at physiological pH and temperature. Adult human bone marrow-derived stem cells (hBMSC) encapsulated in such hydrogels at chitosan/collagen ratios of 100/0, 65/35, 25/75, and 0/100 wt% exhibited high viability at day 1 after encapsulation, but DNA content dropped by about half over 12 days in pure chitosan materials while it increased twofold in materials containing collagen. Collagen-containing materials compacted more strongly and were significantly stiffer than pure chitosan gels. In monolayer culture, exposure of hBMSC to β-GP resulted in decreased cell metabolic activity that varied with concentration and exposure time, but washing effectively removed excess β-GP from hydrogels. The presence of chitosan in materials resulted in higher expression of osterix and bone sialoprotein genes in medium with and without osteogenic supplements. Chitosan also increased alkaline phosphatase activity and calcium deposition in osteogenic medium. Chitosan–collagen composite materials have potential as matrices for cell encapsulation and delivery, or as in situ gel-forming materials for tissue repair.
Keywords: Chitosan; Collagen; Hydrogel; Osteogenic; Tissue engineering; Composite;

Mineralization behavior with mesenchymal stromal cells in a biomimetic hyaluronic acid-based scaffold by Cristina Manferdini; Vincenzo Guarino; Nicoletta Zini; Maria Grazia Raucci; Andrea Ferrari; Francesco Grassi; Elena Gabusi; Stefano Squarzoni; Andrea Facchini; Luigi Ambrosio; Gina Lisignoli (3986-3996).
A biomimetic hyaluronic acid (HA)-based polymer scaffold was analysed in vitro for its characteristics and potential to support mineralization as carrier-vehicle. Biomimetic apatite crystal nucleation on the scaffold surface was obtained by a fine control of the pH level that increased ionic solubility thus controlling apatite formation kinetic. Different concentrations of human mesenchymal stromal cells (h-MSCs) were seeded on the scaffold, osteogenesis was induced in the presence or absence of fibroblast growth factor -2 and mineralization was analysed at different time points. We found that only at the highest h-MSCs concentration tested, the cells were uniformly distributed inside and outside the scaffold and proliferation started to decrease from day 7. Electron microscopy analysis evidenced that h-MSCs produced extracellular matrix but did not establish a direct contact with the scaffold. We found mineralized calcium-positive areas mainly present along the backbone of the scaffold starting from day 21 and increasing at day 35. FGF-2 treatment did not accelerate or increase mineralization. Non-biomimetic HA-based control scaffold showed immature mineralized areas only at day 35. Our data demonstrate that the biomimetic treatment of an HA-based scaffold promotes a faster mineralization process suggesting its possible use in clinics as a support for improving bone repair.
Keywords: Biomimetic material; Hyaluronic acid; SBF; Fibroblast growth factor; Osteogenesis;

The effect of incorporation of SDF-1α into PLGA scaffolds on stem cell recruitment and the inflammatory response by Paul T. Thevenot; Ashwin M. Nair; Jinhui Shen; Parisa Lotfi; Cheng-Yu Ko; Liping Tang (3997-4008).
Despite significant advances in the understanding of tissue responses to biomaterials, most implants are still plagued by inflammatory responses which can lead to fibrotic encapsulation. This is of dire consequence in tissue engineering, where seeded cells and bioactive components are separated from the native tissue, limiting the regenerative potential of the design. Additionally, these interactions prevent desired tissue integration and angiogenesis, preventing functionality of the design. Recent evidence supports that mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) can have beneficial effects which alter the inflammatory responses and improve healing. The purpose of this study was to examine whether stem cells could be targeted to the site of biomaterial implantation and whether increasing local stem cell responses could improve the tissue response to PLGA scaffold implants. Through incorporation of SDF-1α through factor adsorption and mini-osmotic pump delivery, the host-derived stem cell response can be improved resulting in 3X increase in stem cell populations at the interface for up to 2 weeks. These interactions were found to significantly alter the acute mast cell responses, reducing the number of mast cells and degranulated mast cells near the scaffold implants. This led to subsequent downstream reduction in the inflammatory cell responses, and through altered mast cell activation and stem cell participation, increased angiogenesis and decreased fibrotic responses to the scaffold implants. These results support that enhanced recruitment of autologous stem cells can improve the tissue responses to biomaterial implants through modifying/bypassing inflammatory cell responses and jumpstarting stem cell participation in healing at the implant interface.
Keywords: Mesenchymal stem cell; Hematopoietic stem cells; Inflammation; Angiogenesis; Scaffold; Biocompatibility;

The prevention of peritendinous adhesions by a phospholipid polymer hydrogel formed in situ by spontaneous intermolecular interactions by Noriyuki Ishiyama; Toru Moro; Kazuhiko Ishihara; Takashi Ohe; Toshiki Miura; Tomohiro Konno; Tadashi Ohyama; Mizuna Kimura; Masayuki Kyomoto; Kozo Nakamura; Hiroshi Kawaguchi (4009-4016).
Preventing peritendinous adhesions after surgical repair of tendon is difficult. In order to establish an ideal anti-adhesion material, we prepared a spontaneously forming hydrogel by mixing the aqueous solutions of two polymers, poly(MPC-co-methacrylic acid) (PMA) and amphiphilic poly(MPC-co-n-butyl methacrylate) (PMB), in the presence of Fe3+. This PMA/PMB/Fe3+ hydrogel (MPC polymer hydrogel) had a honeycomb microstructure with nanometer-scale pores, which resist cell invasion but allow the passage of cytokines and growth factors for tendon healing. The dissociation rate of the hydrogel could be controlled by changing Fe3+ concentration, and by examining the viscoelasticity of the hydrogel, we determined the optimal Fe3+ concentration to be 0.05 m. We then examined the effects of the in situ application of this MPC polymer hydrogel containing 0.05 m Fe3+ by using two animal models: the rat Achilles tendon model and the chicken flexor digitorum profundus tendon model. In both models, macroscopic and histological observation revealed that peritendinous adhesions were significantly decreased by the hydrogel application. Mechanical analyses revealed that the hydrogel prevented peritendinous adhesions but did not affect the tendon healing. Because of its characteristic microstructure and excellent biocompatibility, we believe that the MPC polymer hydrogel will be ideal for preventing peritendinous adhesions.
Keywords: Anti-adhesion; Tendon; Phospholipid polymer; Hydrogel; Biocompatibility;

In situ endothelialization of intravascular stents coated with an anti-CD34 antibody functionalized heparin–collagen multilayer by Quankui Lin; Xin Ding; Fuyu Qiu; Xiaoxiao Song; Guosheng Fu; Jian Ji (4017-4025).
The in-stent restenosis (ISR) and the late stent thrombosis (LAST) represent the most common failures of stent implantation and are both mediated at the injured endothelium. The natural endothelium healing mechanism provides an approach to achieve in situ endothelialization of the implant by stimulating the neighboring endothelial cells (ECs) migration or capturing the circulating endothelial cells (CEC) directly from the blood circulation. An anti-CD34 antibody functionalized multilayer of heparin/collagen is developed here via layer-by-layer assemble. The ellipsometry and QCM-D results demonstrate that the multilayer coatings with slight glutaraldehyde cross-linking are stable in static incubation and flushing conditions, respectively. The in vitro hemocompatibility tests and cell culture results indicate that both heparin/collagen multilayers with or without the anti-CD34 antibody functionalization not only preserve good hemocompatibility, but also promote cell attachment and growth notably. While the heparin/collagen multilayer coatings show no selectivity in promotion of ECs and smooth muscle cells (SMCs), the anti-CD34 antibody functionalized heparin/collagen multilayers can specifically promote the attachment and growth of the vascular ECs. The metabolic activity assessment and the NO secretion measurements further indicate that the adherent ECs on the anti-CD34 antibody functionalized heparin/collagen multilayer surface have better viability and possess the specific function of the natural vascular ECs. In vivo experiments indicate that the anti-CD34 antibody can enrich and accelerate the attachment of the vascular cells onto the stent and rapid endothelialization is realized. While no significant difference of neointimal hyperplasia is observed between the bare metal stents and heparin/collagen multilayer modified stents, the neointimal hyperplasia on the anti-CD34 antibody functionalized multilayer modified stents is significantly inhibited. The success of the anti-CD34 antibody functionalized heparin/collagen multilayer coating in rapid endothelialization and anti-restenosis might indicate that the immobilization of ECs specific ligand onto a cytocompatible matrix can be a good approach for in situ endothelialization and a possible solution to ISR.
Keywords: Layer-by-layer; Endothelialization; ECs selectivity; Stent;

The in vivo performance of polyvinyl alcohol macro-encapsulated islets by Zhi Qi; Yanna Shen; Goichi Yanai; Kaichiang Yang; Yasumasa Shirouzu; Akihito Hiura; Shoichiro Sumi (4026-4031).
Islet transplantation is a method for the treatment of type 1 diabetes mellitus (DM) and has been widely performed around the world. The long-term cryopreservation of islets shows many advantages in the field of islet transplantation. Previous studies have described the development of sheet-type polyvinyl alcohol (PVA) macro-encapsulated islets (MEI) to treat type 1 DM without any immunotherapy. The present study examined their beneficial effects on islet cryopreservation. PVA MEI of Wistar rats were divided into three groups of 1-day, 7-day and 30-day cryopreservation at −80 °C. The 30-day group showed a lower recovery rate of the islet number and impaired insulin release in comparison to the 1-day group, whereas no significant differences of the in vitro results were observed between the 1-day and 7-day groups. The MEI transplantation recipient mice in the 1-day and 7-day groups reached normoglycemia for a 4-week observation period, and the recipients in 30-day group also showed a significant decrease followed by a slightly higher non-fasting blood glucose level. These results suggest that the PVA MEI are useful for islet long-term cryopreservation, and that the use of cryopreserved PVA MEI may, therefore, be a promising modality for performing DM therapy.
Keywords: Polyvinyl alcohol (PVA); Macro-encapsulated islets (MEIs); Islet cryopreservation; Islet transplantation;

This paper proposes an approach for increasing drug release durations from contact lenses and other biomedical devices by in situ creation of transport barriers of Vitamin E that force drug molecules to diffuse through long tortuous path. Results show that the increase in release duration is quadratic in Vitamin E loading, which is consistent with proposed mathematical models. Loadings of 10 and 40% Vitamin E increase release time of timolol by a factor of about 5 and 400, respectively for NIGHT&DAY™ lens. Similar results have been obtained for other hydrophilic drugs including fluconazole and dexamethasone 21-disodium phosphate (DXP). Vitamin E loading in the NIGHT&DAY™ lens leads to slight increase in lens sizes (6.5% increase for 30% loading), a slight reduction in oxygen diffusion (about 40% reduction for 75% loading), and a more significant reduction in the ion permeability (50% reduction for 10% loading). Additionally, Vitamin E loading has a beneficial effect of blocking UV radiation which will reduce the corneal damage due to UV light.
Keywords: Silicone contact lens; Vitamin E; Diffusion barriers; Extended release; Ophthalmic drug delivery;

A poly(propylene fumarate) – Calcium phosphate based angiogenic injectable bone cement for femoral head osteonecrosis by Chih-Hung Chang; Tai-Chieh Liao; Yuan-Ming Hsu; Hsu-Wei Fang; Chia-Chun Chen; Feng-Huei Lin (4048-4055).
Osteonecrosis of the femoral head commonly occurs when the blood supply to bone was disrupted. The general treatment for early stages of necrosis in the femoral head is core decompression. However, the long-term outcome of this operation is usually compromised due to collapse of the necrotic bone. In this study, poly(propylene fumarate) (PPF) and calcium phosphate cement (CPC) were combined to provide appropriate mechanical strength after core-decompressed femoral heads and offer the properties of osteoconductivity. Effects of different ratios of CPC to PPF on mechanical and cytotoxicity were investigated. Results show that bone cement is less cytotoxic with the C/P ratio raise, and the increment of the CPC proportion also strengthens the mechanical strength, reduces the crosslinking temperature and diminishes excessive swelling of the cement. With addition of ginsenoside Rg1 the bone cement composite can also offer angiogenic effect. The drug release profiles were analyzed and the angiogenecity of released Rg1 was confirmed by the assay of tube formation in human umbilical vein endothelial cells (HUVECs). In summary, the newly developed angiogenic bone cement composite possesses remarkable development potential for application to treating osteonecrosis of the femoral head.
Keywords: Injectable bone cement; Angiogenesis; Poly(propylene fumarate); Calcium phosphate; Ginsenoside Rg1;

A polydioxanone electrospun valved patch to replace the right ventricular outflow tract in a growing lamb model by David Kalfa; Alain Bel; Annabel Chen-Tournoux; Alberto Della Martina; Philippe Rochereau; Cyrielle Coz; Valérie Bellamy; Mourad Bensalah; Valérie Vanneaux; Séverine Lecourt; Elie Mousseaux; Patrick Bruneval; Jérôme Larghero; Philippe Menasché (4056-4063).
A major issue in congenital heart surgery is the lack of viable right ventricular outflow tract (RVOT) replacement materials. Several biomaterials have been used, with different scaffolds and cells, but they have failed to restore a tri-layered RVOT, and reoperations are often required. We investigated the function, histological changes and potential of growth and tissue regeneration of polydioxanone (PDO) electrospun bioabsorbable valved patches seeded with mesenchymal stem cells (MSCs) in the RVOT of growing lambs. Autologous blood-derived MSCs were labeled with quantum dots and seeded on PDO electrospun valved patches. Those were implanted into the RVOT of 6 growing lambs followed up until 8 months. Results were assessed by echocardiography, magnetic resonance imaging (MRI), histology, immunohistochemistry and biochemical assays. Tissue-engineered RVOT were neither stenotic nor aneurismal and displayed a growth potential, with less fibrosis, less calcifications and no thrombus compared with control polytetrafluoroethylene (PTFE)-pericardial patches. The PDO scaffold was completely degraded and replaced by a viable, three-layered, endothelialized tissue and an extracellular matrix with elastic fibers similar to that of native tissue. Detection of quantum dots at 1 month suggested that at least some of the cells were-derived from the grafted cells. A polydioxanone electrospun tissue-engineered valved transannular patch seems to be a promising device in restoring a living RVOT and could ultimately lead to applications in the treatment of congenital RVOT diseases.
Keywords: Polydioxanone; Cardiac tissue engineering; Mesenchymal stem cells; ECM (extracellular matrix); Biodegradation;

The ability of corneal epithelial cells to recognize high aspect ratio nanostructures by Elizabeth J. Tocce; Valery K. Smirnov; Dmitry S. Kibalov; Sara J. Liliensiek; Christopher J. Murphy; Paul F. Nealey (4064-4072).
The basement membrane of the human corneal epithelium comprises topographic features including fibers, pores, and elevations with feature dimensions on the order of 20–400 nm. Understanding the impact of sub-micron and nanotopography on corneal cell behavior will contribute to our understanding of biomechanical cues and will assist in the design of improved synthetic corneal implants. We utilized well defined ridge and groove wave-like nanostructures (wave ordered structures, WOS) of 60–140 pitches (30–70 nm ridge widths) and 200 nm depths to assess human corneal epithelial cell (HCEC) contact guidance and to establish HCEC contact acuity defined as the lower limit in feature dimensions at which cells respond to biomimetic topographic cues. Results using the WOS substrates demonstrate that HCEC contact acuity is in the range of 60 nm pitch for cells in a serum-free basal medium (EpiLife®) and in the range of 90 nm pitch for cells in epithelial medium. To further investigate the influence of HCEC contact acuity in the presence of larger topographic cues, we fabricated 70 nm pitch WOS-overlaid parallel to the top of the ridges of 800–4000 nm pitch. HCEC cultured in epithelial medium demonstrate a significant increase in the percent of cells aligning to 4000 nm pitch topography with WOS-overlay compared to controls (both flat and 70 nm WOS alone) and 4000 nm pitch topography alone. These results highlight the significance of the lower range of basement membrane scale topographic cues on cell response and allow for improved prosthetic design.
Keywords: Cornea; Epithelial cell; Nanotopography; Contact guidance;

There is continuous interest in developing manganese-based T 1 contrast agents. While much effort has been made to synthesize manganese chelates, the development of manganese-based nanoparticle, particularly manganese oxides, as MRI contrast agents is burgeoning. In this report, sub-10-nm nanospheres, nanoplates, and nanocubes of Mn3O4 were synthesized and exhibited paramagnetic behavior at room temperature on the basis of superconducting quantum interference devices (SQUID) measurements. The surface Mn3+ passivated nanoplates examined by x-ray photoelectron spectroscopy (XPS) had the largest r 1 relaxivity of the reported manganese oxide nanoparticles. The MR labeling assays of Mn3O4 nanoplate-treated A549 lung cancer cells showed that MR signals increased to 139% in T 1-weighted images compared with untreated cells when the Mn ion concentration went down to 1.3 × 10−2 mm. A dark field illumination microscope was employed to monitor Mn3O4 nanoplates internalized into cells as a function of time.
Keywords: Mn3O4; Nanoparticles; MRI and contrast agents;

Bactericidal and virucidal ultrathin films assembled layer by layer from polycationic N-alkylated polyethylenimines and polyanions by Sze Yinn Wong; Qing Li; Jovana Veselinovic; Byeong-Su Kim; Alexander M. Klibanov; Paula T. Hammond (4079-4087).
In this work, we designed contact-killing ionically cross-linked polymeric thin films using Layer-by-Layer (LbL) technology. A polycation, N,N-dodecyl,methyl-polyethylenimine, with microbicidal activity was layered with a polyanion, such as poly(acrylic acid), to create LbL films highly effective against both airborne and waterborne Escherichia coli and Staphylococcus aureus (Gram negative and positive bacteria, respectively), as well as influenza A/WSN (H1N1) virus. The dependence of the microbicidal activity on the pH during and post-assembly of LbL film formation, the nature of the polycation and polyanion, the number of layers in the LbL film, and other experimental variables was investigated quantitatively.
Keywords: Layer-by-Layer (LbL); Polyelectrolyte multilayer; Microbicidal; Bactericidal; Virucidal; Contact-killing;

Near infrared photoacoustic detection of sentinel lymph nodes with gold nanobeacons by Dipanjan Pan; Manojit Pramanik; Angana Senpan; Soumojit Ghosh; Samuel A. Wickline; Lihong V. Wang; Gregory M. Lanza (4088-4093).
Detection of sentinel lymph node (SLN) using photoacoustic imaging is an emerging technique for noninvasive axillary staging of breast cancer. Due to the absence of intrinsic contrast inside the lymph nodes, exogenous contrast agents are used for photoacoustic detection. In this work, we have demonstrated near infrared detection of SLN with gold nanobeacons (GNBs) providing the photoacoustic contrast in a rodent model. We found that size dictates the in vivo characteristics of these nanoparticles in SLN imaging. Larger nanobeacons with high payloads of gold were not as efficient as smaller size nanobeacons with lower payloads for this purpose. Colloidal GNBs were designed as a nanomedicine platform with “soft” nature that is amenable to bio-elimination, an essential feature for in vivo efficacy and safety. The GNBs were synthesized as lipid- or polymer-encapsulated colloidal particles incorporating tiny gold nanoparticles (2–4 nm) in three tunable sizes (90 nm, 150 nm and 290 nm). Smaller GNBs were noted trafficking through the lymphatic system and accumulating more efficiently in the lymph nodes in comparison to the bigger nanoagents. At 20 min, the GNBs reached the SLN and were no longer observed within the draining lymphatic vessel. Within 1 h post-injection, the contrast ratio of the lymph nodes with the surrounding blood vessels was 9:1. These findings were also supported by analytical measurements of the ex vivo tissue samples. Results indicate that cumulative nanoparticle deposition in lymph nodes is size dependent and that high payloads of gold, although offering greater contrast in vitro, may yield nanoagents with poor intradermal migration and lymphatic transport characteristics.
Keywords: Gold nanoparticle; Near infrared imaging; Photoacoustic imaging; Sentinel lymph node detection; Breast cancer staging;

Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells by Hiroshi Yukawa; Yukimasa Kagami; Masaki Watanabe; Koichi Oishi; Yoshitaka Miyamoto; Yukihiro Okamoto; Manabu Tokeshi; Noritada Kaji; Hirofumi Noguchi; Kenji Ono; Makoto Sawada; Yoshinobu Baba; Nobuyuki Hamajima; Shuji Hayashi (4094-4103).
Quantum dots (QDs) have been used to study the effects of fluorescent probes for biomolecules and cell imaging. Adipose tissue-derived stem cells, which carry a relatively lower donor site morbidity, while yielding a large number of stem cells at harvest, were transduced with QDs using the octa-arginine peptide (R8) cell-penetrating peptide (CPP). The concentration ratio of QDs:R8 of 1 × 104 was optimal for delivery into ASCs. No cytotoxicity was observed in ASCs transduced with less than 16 nM of QDs655. In addition, >80% of the cells could be labeled within 1 h and the fluorescent intensity was maintained at least for 2 weeks. The ASCs transduced with QDs using R8 could be differentiated into both adipogenic and osteogenic cells, thus suggesting that the cells maintained their stem cell potency. The ASCs labeled with QDs using R8 were further transplanted subcutaneously into the backs of mice or into mice through the tail vein. The labeled ASCs could be imaged with good contrast using the Maestro in vivo imaging system. These data suggested that QD labeling using R8 could be utilized for the imaging of ASCs.
Keywords: Semiconductor; Quantum dots (QDs); Cell-penetrating peptides (CPPs); In vivo imaging; Adipose tissue-derived stem cells (ASCs);

In situ real-time investigation of cancer cell photothermolysis mediated by excited gold nanorod surface plasmons by Cheng-Lung Chen; Ling-Ru Kuo; Ching-Lin Chang; Yeu-Kuang Hwu; Cheng-Kuang Huang; Shin-Yu Lee; Kowa Chen; Su-Jien Lin; Jing-Duan Huang; Yang-Yuan Chen (4104-4112).
The photothermolysis of living EMT-6 breast tumor cells triggered by gold nanorods (AuNRs) with two-photon irradiation was conducted in situ and under real-time observation. The morphology and plasma membrane permeability of the cells were key indicators to the phenomena. AuNRs with an aspect ratio of 3.92, and a longitudinal absorption peak at 800 nm were synthesized with a seed-mediated method. The nanorods surfaces were further modified with polystyrenesulfonate (PSS) for biocompatibility. The prepared nanorods displayed excellent two-photon photoluminescence imaging. In situ real-time results revealed cavities internal to the cells were created from thermal explosions triggered by AuNRs localized photothermal effect. The cavitation dynamic is energy dependent and responsible for the perforation or sudden rupture of the plasma membrane. The energy threshold for cell therapy depended significantly on the number of nanorods taken up per cell. For an ingested AuNR cluster quantity N ∼ 10–30 per cell, it is found that energy fluences E larger-than 93 mJ/cm2 led to effective cell destruction in the crumbled form within a very short period. As for a lower energy level E = 18 mJ/cm2 with N ∼ 60–100, a non-instant, but progressive cell deterioration, is observed.
Keywords: Gold; Plasma; Membrane; Laser; Fluorescence;

An anisotropic nanofiber/microsphere composite with controlled release of biomolecules for fibrous tissue engineering by Lara C. Ionescu; Gregory C. Lee; Brian J. Sennett; Jason A. Burdick; Robert L. Mauck (4113-4120).
Aligned nanofibrous scaffolds can recapitulate the structural hierarchy of fiber-reinforced tissues of the musculoskeletal system. While these electrospun fibrous scaffolds provide physical cues that can direct tissue formation when seeded with cells, the ability to chemically guide a population of cells, without disrupting scaffold mechanical properties, would improve the maturation of such constructs and add additional functionality to the system both in vitro and in vivo. In this study, we developed a fabrication technique to entrap drug-delivering microspheres within nanofibrous scaffolds. We hypothesized that entrapping microspheres between fibers would have a less adverse impact on mechanical properties than placing microspheres within the fibers themselves, and that the composite would exhibit sustained release of multiple model compounds. Our results show that microspheres ranging from 10 ∼ 20 microns in diameter could be electrospun in a dose-dependent manner to form nanofibrous composites. When delivered in a sacrificial PEO fiber population, microspheres remained securely entrapped between slow-degrading PCL fibers after removal of the sacrificial delivery component. Stiffness and modulus of the composite decreased with increasing microsphere density for composites in which microspheres were entrapped within each fiber, while stiffness did not change when microspheres were entrapped between fibers. The release profiles of the composite structures were similar to free microspheres, with an initial burst release followed by a sustained release of the model molecules over 4 weeks. Further, multiple model molecules were released from a single scaffold composite, demonstrating the capacity for multi-factor controlled release ideal for complex growth factor delivery from these structures.
Keywords: Mechanical properties; Drug delivery; Microsphere; Nanospun scaffold;

Long acting hyaluronate – exendin 4 conjugate for the treatment of type 2 diabetes by Ji-Hyun Kong; Eun Ju Oh; Su Young Chae; Kang Choon Lee; Sei Kwang Hahn (4121-4128).
Despite clinical exploitation of exendin 4 for the treatment of type 2 diabetes, the significantly short half-life requiring twice a day injection has limited the wide applications. In this work, a protocol for the synthesis of long acting hyaluronate (HA) – exendin 4 conjugate was successfully developed using Michael addition chemistry between vinyl sulfone modified HA (HA-VS) and thiolated exendin 4. The exendin 4 content could be controlled in the range of 5–30 molecules per single HA chain with a bioconjugation efficiency higher than 90%. The conjugation of exendin 4 with HA resulted in about 20 times improved in vitro serum stability maintaining the hypoglycemic and gluco-regulatory bioactivities of exendin 4. HA – exendin 4 conjugates showed excellent glucose-lowering capabilities in type 2 db/db mice demonstrating protracted hypoglycemic effect up to 3 days after a single subcutaneous injection. Furthermore, insulin immunohistochemical analysis of islets in db/db mice confirmed the improved insulinotropic activity of HA – exendin 4 conjugates. The HA – exendin 4 conjugates will be investigated further as a twice a week injection dosage form for clinical applications.
Keywords: Hyaluronic acid; Exendin-4; Conjugation; Drug delivery; Type 2 diabetes;

Folate-PEG coated cationic modified chitosan – Cholesterol liposomes for tumor-targeted drug delivery by Hanjie Wang; Peiqi Zhao; Xiaofei Liang; Xiaqun Gong; Tao Song; Ruifang Niu; Jin Chang (4129-4138).
In this paper, a folate-PEG coated polymeric liposome (FPL) formed from octadecyl-quaternized lysine modified chitosan (OQLCS) and cholesterol has been prepared successfully. The OQLCS and its derivatives were characterized using 1H NMR and infrared spectrum analysis. The FPLs properties were extensively studied by dynamic light scattering (DLS), fluorescence spectroscopy, and transmission electron microscopy (TEM). Due to the amphiphilic property and positive zeta potential of OQLCS, the OQLCS and cholesterol can form stable core-shell FPLs with small size (effective diameter: 163.5 nm) and narrow distribution (polydispersity: 0.108) in aqueous solutions. The PLs could form multi-lamellar structure similar to that of traditional liposomes prepared from phosphatidylcholine/cholesterol (PC/Chol). Compared with traditional liposome, calcein-loaded Polymeric Liposome exhibited high encapsulation efficiency in aqueous solution and slow, controlled release under different pH conditions. Most important, in cellular uptake experiment, folate coated FPLs showed significant higher uptake by MCF-7 cells as compared to FPLs without folate and traditional liposomes, because of the folate-receptor mediated endocytosis. The data suggest that the folate-PEG coated polymeric liposomes (FPLs) may be a useful drug delivery system.
Keywords: Chitin/chitosan; Controlled drug release; Drug delivery; Nanoparticle; Liposome; Cancer nanotechnology;

Development of a bifunctional immunoliposome system for combined drug delivery and imaging in vivo by Bin Feng; Kazuhito Tomizawa; Hiroyuki Michiue; Xiao-Jian Han; Shin-ichi Miyatake; Hideki Matsui (4139-4145).
The diverse characteristics of immunoliposomes provide advantages for utilization in drug delivery systems. In this study, we fused the antibody affinity motif of protein A (ZZ) with Gaussia luciferase (GLase). The fused protein conjugated with an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (GLase-ZZ-His-mAb) was effectively delivered into glioma cells expressing an activated EGFR mutant (EGFRvIII) and the bioluminescence was visualized in the cells. Immunoliposomes were further constructed with DSPE-PEG-MAL for covalent GLase-ZZ-His-mAb conjugation. A fluorescence dye (HPTS) encapsulated in immunoliposomes conjugated with GLase-ZZ-His-mAb was effectively delivered into EGFRvIII-expressing glioma cells. In a murine xenograft model of glioma, moreover, specific targeting of the immunoliposomes was visualized in the tumor. This new bifunctional immunoliposome system has the potential for drug delivery and imaging in vivo.
Keywords: Glioma cells; EGFR; Immunoliposome; Gaussia luciferase; Bioluminescence; Boron neutron capture therapy;

Antibiotic-releasing porous polymethylmethacrylate constructs for osseous space maintenance and infection control by Meng Shi; James D. Kretlow; Anson Nguyen; Simon Young; L. Scott Baggett; Mark E. Wong; F. Kurtis Kasper; Antonios G. Mikos (4146-4156).
The use of a strategy involving space maintenance as the initial step of a two-stage regenerative medicine approach toward reconstructing significant bony or composite tissue defects in the craniofacial area, preserves the void volume of bony defects and could promote soft tissue healing prior to the subsequent definitive repair. One of the complications with a biomaterial-based space maintenance approach is local infection, which requires early, effective eradication, ideally through local antibiotic delivery. The purpose of this study is to develop a dual function implant material for maintaining osseous space and releasing an antibiotic to eliminate local infection in bony defects. Colistin, a polymyxin antibiotic, was chosen specifically to address infections with Acinetobacter species, the most common pathogen associated with combat-related traumatic craniofacial injuries. Porous polymethylmethacrylate (PMMA) constructs incorporating poly(lactic-co-glycolic acid) (PLGA) microspheres were fabricated by mixing a clinically used bone cement formulation of PMMA powder and methylmethacrylate liquid with a carboxymethylcellulose (CMC) hydrogel (40 or 50 wt%) to impart porosity and PLGA microspheres (10 or 15 wt%) loaded with colistin to control drug release. The PMMA/CMC/PLGA construct featured mild setting temperature, controllable surface/bulk porosity by incorporation of the CMC hydrogel, reasonably strong compressive properties, and continuous drug release over a period of 5 weeks with total drug release of 68.1–88.3%, depending on the weight percentage of CMC and PLGA incorporation. The concentration of released colistin was well above its reported minimum inhibitory concentration against susceptible species for 5 weeks. This study provides information on the composition parameters that enable viable porosity characteristics/drug release kinetics of the PMMA/CMC/PLGA construct for the initial space maintenance as part of a two-stage regenerative medicine approach.
Keywords: Bone tissue engineering; Scaffolds; Biodegradable microspheres; Polymethylmethacrylate; Carboxymethylcellulose; Colistin;

A chitosan/β-glycerophosphate thermo-sensitive gel for the delivery of ellagic acid for the treatment of brain cancer by Sungwoo Kim; Satoru K. Nishimoto; Joel D. Bumgardner; Warren O. Haggard; M. Waleed Gaber; Yunzhi Yang (4157-4166).
We report here the development of a chitosan/β-glycerophosphate(Ch/β-GP) thermo-sensitive gel to deliver ellagic acid (EA) for cancer treatment. The properties of the Ch/β-GP gels were characterized regarding chemical structure, surface morphology, and viscoelasticity. In vitro EA release rate from the EA loaded Ch/β-GP gel and chitosan degradation rate were investigated. The anti-tumor effect of the EA loaded Ch/β-GP gel on brain cancer cells (human U87 glioblastomas and rat C6 glioma cells) was evaluated by examining cell viability. Cell number and activity were monitored by the MTS assay. The Ch/β-GP solution formed a heat-induced gel at body temperature, and the gelation temperature and time were affected by the final pH of the Ch/β-GP solution. The lysozyme increased the EA release rate by 2.5 times higher than that in the absence of lysozyme. Dialyzed chitosan solution with final pH 6.3 greatly reduced the β-GP needed for gelation, thereby significantly improving the biocompatibility of gel (p < 0.001). The chitosan gels containing 1% (w/v) of ellagic acid significantly reduced viability of U87 cells and C6 cells compared with the chitosan gels at 3 days incubation (p < 0.01, and p < 0.001, respectively).
Keywords: Cancer; Injectable gel; Chitosan; Ellagic acid; β-Glycerophosphate (β-GP); Biocompatibility;

Red blood cell membrane grafting of multi-functional hyperbranched polyglycerols by Nicholas A.A. Rossi; Iren Constantinescu; Rajesh K. Kainthan; Donald E. Brooks; Mark D. Scott; Jayachandran N. Kizhakkedathu (4167-4178).
The covalent attachment of hydrophilic polymers or biopharmaceuticals to the surface of red blood cells (RBCs) has previously been shown as a relatively compatible and effective method for a range of applications. Here, the first example of cell-surface grafting with a hyperbranched and multi-functional macromolecule is described. A range (3 kDa–101 kDa) of dense, globular, and blood compatible hyperbranched polyglycerols (HPG) were synthesized and functionalized with cell-surface reactive, succinimidyl succinate groups (1–12 groups per polymer). Subsequently, HPG was grafted to the RBCs, which were analyzed using physical characterization techniques such as aqueous two-phase partitioning and particle electrophoresis. It was found that the extent of grafting was enhanced by increasing HPG molecular weight, the number of reactive groups per HPG, HPG concentration, and reaction time. Good in vitro cell viability – as measured by lipid peroxidation, hemoglobin oxidation, cell lysis, osmotic fragility, stability in fresh serum and aggregation behavior – was observed for grafting concentrations up to 4.8 mm. The multi-functional aspect of HPG is highlighted by the following observations: using fluorescein-labeled Anti-D (monoclonal) antibody and flow cytometry, the detection of cell-surface Rhesus (RhD) antigens were significantly reduced upon HPG grafting. Secondly, the potential for using HPG as a multi-functional, delivery agent was demonstrated by attaching fluorescent markers to the HPG via degradable linkages prior to grafting.
Keywords: Red blood cell surface modification; Multi-functional polymer grafting; Hyperbranched polyglycerol; Antigen masking; Fluorescently-labeled polymers;

Relationship between the tautomeric structures of curcumin derivatives and their Aβ-binding activities in the context of therapies for Alzheimer's disease by Daijiro Yanagisawa; Nobuaki Shirai; Tomone Amatsubo; Hiroyasu Taguchi; Koichi Hirao; Makoto Urushitani; Shigehiro Morikawa; Toshiro Inubushi; Masanari Kato; Fuminori Kato; Kyuya Morino; Hirohiko Kimura; Ichiro Nakano; Chikako Yoshida; Takashi Okada; Mitsuo Sano; Yoshiko Wada; Ken-nosuke Wada; Akitsugu Yamamoto; Ikuo Tooyama (4179-4185).
Curcumin, which can exist in an equilibrium between keto and enol tautomers, binds to β-amyloid (Aβ) fibrils/aggregates. The aim of this study was to assess the relationship between the tautomeric structures of curcumin derivatives and their Aβ-binding activities. Curcumin derivatives with keto-enol tautomerism showed high levels of binding to Aβ aggregates but not to Aβ monomers. The binding activity of the keto form analogue of curcumin to Aβ aggregates was found to be much weaker than that of curcumin derivatives with keto-enol tautomerism. The color of a curcumin derivative with keto-enol tautomerism, which was substituted at the C-4 position, changed from yellow to orange within 30 min of being combined with Aβ aggregates in physiological buffer. This resulted from a remarkable increase in the enol form with extended conjugation of double bonds upon binding. These findings suggest that curcumin derivatives exist predominantly in the enol form during binding to Aβ aggregates, and that the enolization of curcumin derivatives is crucial for binding to Aβ aggregates. The keto-enol tautomerism of curcumin derivatives may be a novel target for the design of amyloid-binding agents that can be used both for therapy and for amyloid detection in Alzheimer's disease.
Keywords: Alzheimer's disease; Amyloid-β; Curcumin; Keto-enol tautomerism; Amyloid detection;

Cationic polymers are widely studied as gene-delivery vehicles, but are limited by low transfection due to inhibited release of DNA, and high cytotoxicity from the requisite positive charges. Here, we introduce a hydrolytic cationic ester polymer containing both tertiary and quaternary amines, which packages DNA into nanoparticles and then releases DNA upon hydrolysis. Cells were transfected with these nanoparticles. Luciferase expression from a polymer with the tertiary/quaternary ratio of 1:1 was equal to that obtained using branched polyethylenimine (PEI), and expression from an acidified polymer with the ratio of 3:1 was 20 times higher than branched PEI. These ratios best balance proton sponging from tertiary amines and packaging ability from cations. Importantly, no hydrolysed polymer exhibited cytotoxicity; the zwitterionic nature of the hydrolysed polymer ensured that the quaternary amines in this work do not cause cell death. Hydrolysis is critical for effective and safe gene therapy.
Keywords: Gene transfer; Cell viability; Hydrolysis; Biocompatibility;

Polycation/DNA complexes coated with oligonucleotides for gene delivery by Yi-Chen Chung; Wen-Yuan Hsieh; Tai-Horng Young (4194-4203).
Ternary nanoparticles with negatively charged surface were prepared by coating single-stranded oligonucleotides (5′–C10A20–3′) on histidine-conjugated polyallylamine (PAA-HIS)/DNA complexes for gene delivery. Characterization of PAA-HIS/DNA/oligonucleotide complexes demonstrated that nanoparticles possessed the negative surface charge −27 mV and size of around 100 nm when the molar ratio of oligonucleotide/PAA-HIS exceeded 1.5. The negatively charged oligonucleotide-coated PAA-HIS/DNA complexes could be entirely internalized by the living HeLa cells to exhibit high gene expression with low cytotoxicity and the resistance against erythrocyte agglutination and serum inhibition. Since the gene expression of PAA-HIS/DNA complexes was significantly inhibited by coating other polyanions and oligonucleotides, the ternary PAA-HIS/DNA/deoxyadenosine-rich oligonucleotide complexes were uptaken by specific receptor-mediated process. Additionally, the deposition of a layer of oligonucleotides onto the binary PAA-HIS/DNA complexes could effectively transfect various types of cells including HEK-293, HepG2 and Hs68 cells, indicating the technology of coating specific oligonucleotides on PAA-HIS/DNA complexes or other cationic binary DNA complexes might facilitate the use of nanoparticles for safe and efficient gene delivery and eventual therapy.
Keywords: Ternary nanoparticles; Polyallylamine; Histidine; Oligonucleotides; Gene delivery;

Hybrid superparamagnetic iron oxide nanoparticle-branched polyethylenimine magnetoplexes for gene transfection of vascular endothelial cells by Ran Namgung; Kaushik Singha; Mi Kyung Yu; Sangyong Jon; Yong Sook Kim; Youngkeun Ahn; In-Kyu Park; Won Jong Kim (4204-4213).
The work demonstrated the development of thermally cross-linked superparamagnetic nanomaterial which possessed polyethylene glycol moiety and covalently linked branched polyethylenimine (BPEI), and exhibited highly efficient magnetofection even under serum conditioned media. The study showed its high anti-biofouling, cell viability and serum stability and thus revealed a potential magnetic nanoparticle-mediated targeted gene delivery system. This superparamagnetic particle mediated rapid and efficient transfection in primary vascular endothelial cells (HUVEC) successfully inhibits expression of PAI-1 which is responsible for various vascular dysfunctions such as vascular inflammation and atherosclerosis and thereby provides a potential strategy to transfect highly sensitive HUVEC. The sequential steps for the enhanced magnetofection had been studied by monitoring cellular uptake with the aid of confocal microscopy.
Keywords: Magnetofection; Superparamagnetic iron oxide; Polyethylenimine; Gene delivery; Atherosclerosis;

Gene delivery using dimethyldidodecylammonium bromide-coated PLGA nanoparticles by François Fay; Derek J. Quinn; Brendan F. Gilmore; Paul A. McCarron; Christopher J. Scott (4214-4222).
In this present work we describe a poly(lactic-co-glycolic acid) (PLGA) nanoparticle formulation for intracellular delivery of plasmid DNA. This formulation was developed to encapsulate DNA within PLGA nanoparticles that combined salting out and emulsion–evaporation processes. This process reduced the requirement for sonication which can induce degradation of the DNA. A monodispersed nanoparticle population with a mean diameter of approximately 240 nm was produced, entrapping a model plasmid DNA in both supercoiled and open circular structures. To induce endosomal escape of the nanoparticles, a superficial cationic charge was introduced using positively charged surfactants cetyl trimethylammonium bromide (CTAB) and dimethyldidodecylammonium bromide (DMAB), which resulted in elevated zeta potentials. As expected, both cationic coatings reduced cell viability, but at equivalent positive zeta potentials, the DMAB coated nanoparticles induced significantly less cytotoxicity than those coated with CTAB. Fluorescence and transmission electron microscopy demonstrated that the DMAB coated cationic nanoparticles were able to evade the endosomal lumen and localise in the cytosol of treated cells. Consequently, DMAB coated PLGA nanoparticles loaded with a GFP reporter plasmid exhibited significant improvements in transfection efficiencies with comparison to non-modified particles, highlighting their functional usefulness. These nanoparticles may be useful in delivery of gene therapies to targeted cells.
Keywords: Gene therapy; Nanoparticle; Drug delivery; Polyglycolic acid; Polylactic acid; Dimethyldidodecylammonium bromide;

Respiratory syncytial virus (RSV) is a significant cause of morbidity in humans. To date, no effective treatments exist and current prophylactic therapy access is limited and is only ∼50% effective. To attenuate the risk of RSV infection, we hypothesized that bioengineering of either the virus particle or host cell via the covalent grafting of methoxypoly(ethylene glycol) [mPEG] would prevent infection. To this end, the anti-viral effects of grafting concentration, linker chemistry and polymer length on RSV infection was assessed. For viral modification, short chain polymers (2 kDa) were significantly more effective than long chain (20 kDa) polymers. In contrast, modification of host cells with small polymers provided no (∼0%) protection while long chain polymers effectively prevented infection. For example, at 48 hours post-infection at a multiplicity of infection of 0.5 and grafting concentrations of 5, 7.5, and 15 mm, 20 kDa mPEG decreased infection by 45, 83, and 91%, respectively. Importantly, both viral and host cell PEGylation strategies were able to provide near complete protection against RSV infection of both non-polarized and polarized cells. In conclusion, mPEG-modification of either RSV or the host cell is a highly effective prophylactic strategy for preventing viral infection.
Keywords: Antimicrobial; Respiratory syncytial virus (RSV); Polyethylene glycol (polyethylene oxide); Microbiology; Immunomodulation; Infection;

Here, we report that two distinctive cell populations with osteoblastic differentiation ability were found in adherent cell populations from bone marrow. Mesenchymal stem cells (MSCs) were conventionally isolated by using adherent property of bone marrow cells onto a plastic culture dish. MSCs enriched on the basis of their adherent property were considered phenotypically and functionally heterogeneous. We developed a ligand-immobilized surface for separating subpopulation of adherent cells derived from bone marrow by the cell rolling process. We successfully isolate two cell populations with high differentiation ability for osteoblasts in adherent bone marrow cells by using the anti-CD34 antibody-immobilized column. The antibody was covalently conjugated with polyacrylic acid and introduced onto the inner surface of a silicone tube. When cell suspension of MSCs was injected into the antibody-immobilized column, different cell populations were isolated. After the cultivation of isolated cells in the osteoblastic differentiation medium for 1 week, few sub-populations were strongly induced to form osteoblastic cells. This study revealed that the ligand-immobilized surface can be used to continually separate cell populations under a labeling-free condition.
Keywords: Mesenchymal stem cells; Selection; Interface; Osteoblast;

Crack arrest within teeth at the dentinoenamel junction caused by elastic modulus mismatch by Sabine Bechtle; Theo Fett; Gabriele Rizzi; Stefan Habelitz; Arndt Klocke; Gerold A. Schneider (4238-4247).
Enamel and dentin compose the crowns of human teeth. They are joined at the dentinoenamel junction (DEJ) which is a very strong and well-bonded interface unlikely to fail within healthy teeth despite the formation of multiple cracks within enamel during a lifetime of exposure to masticatory forces. These cracks commonly are arrested when reaching the DEJ. The phenomenon of crack arrest at the DEJ is described in many publications but there is little consensus on the underlying cause and mechanism. Explanations range from the DEJ having a larger toughness than both enamel and dentin up to the assumption that not the DEJ itself causes crack arrest but the so-called mantle dentin, a thin material layer close to the DEJ that is somewhat softer than the bulk dentin. In this study we conducted 3-point bending experiments with bending bars consisting of the DEJ and surrounding enamel and dentin to investigate crack propagation and arrest within the DEJ region. Calculated stress intensities around crack tips were found to be highly influenced by the elastic modulus mismatch between enamel and dentin and hence, the phenomenon of crack arrest at the DEJ could be explained accordingly via this elastic modulus mismatch.
Keywords: Dentinoenamel junction; Elastic modulus mismatch; Stress intensity factor; Enamel; Dentin; Toughness;